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,
From the * Department of Urology, West China
Hospital, Sichuan University Sichuan, People's Republic of China; and
Laboratory of Cell Apoptosis & Signaling
Transduction and Laboratory of Oncology, the State Key Laboratory of
Biotherapy, Sichuan University Sichuan, People's Republic of China.
| Correspondence to: Qiao Zhou, Department of Urology, West China Hospital, Sichuan University, 37 Guoxue Xiang St, Chendu, Sichuan, 610041, People's Republic of China (e-mail: kucaizeng{at}163.com). |
| Received for publication January 30, 2007; accepted for publication May 14, 2007. |
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Abstract |
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Key words: Cytosine deaminase, prostate adenocarcinoma
For broad suicide gene therapy, most work has been focused on cytosine
deaminase/5-fluorocytosine (CD/5-FC) and herpes simplex virus thymidine
kinase/gancielduiv (HSV-tk/GCV) systems. The CD gene, an enzyme, present in
fungi and bacteria but absent from mammalian cells, could convert cytosine
into uracil. CD could also deaminate the nontoxic pro-drug 5-FC to its highly
toxic derivative 5-FU, which is then converted by cellular enzymes into 5-FUTP
and 5-Fluoro-dUMP (5-FdUMP). 5-FUTP could be incorporated into RNA to replace
UTP, resulting in the inhibition of the nuclear processing of recombinant and
messenger RNAs (rRNAs and mRNAs), whereas 5-FdUMP could irreversibly inhibit
thymidylate synthetase to prevent DNA synthesis. The CD/5-FC system has been
widely applied in studies of various tumors in the past years because of its
predominantly bystander effect compared with other suicide gene systems
(Ichikawa et al, 2000).
Advanced approaches have utilized regulatory elements of several specific
tumor markers, including prostate-specific antigen (PSA), HSV-tk/GCV
carcinoembryonic antigen (CEA), and 
-fetoprotein (AFP), to achieve
target gene expression in tumor cells exclusively
(Tanaka et al, 1996;
Spitzweg et al, 1999;
Huang et al, 2002).
Several prostatic-specific markers, such as PSA, human glandular kallikrein 2 (hk2), probasin, and prostate androgen-regulatory gene (PAR), have been applied to targeting gene therapy. Results of these experiments showed that these markers could mediate prostate-specific therapeutic effects (Spitzweg et al, 1999; Yu et al, 1999; Xie et al, 2001; Xu et al, 2006), but the prostate-specific regulatory elements mentioned above were down-regulated by androgen deprivation (Israeli et al, 1994), which limited the effect on advanced or metastatic prostate adenocarcinoma. Prostate-specific membrane antigen (PSMA), a new prostate-specific marker, is a type II transmembrane glycoprotein. It has been confirmed to have high expression in advanced or metastatic prostate adenocarcinoma and is positively related to the Gleason score (Wright et al, 1996). PSMA may be a better element for targeting gene therapy in advanced and metastatic prostate adenocarcinomas. O'Keefe et al (2000) reported that they have verified the specific transcriptional activity of the PSMA enhancer/promoter transduced by the plasmid vector, but the efficiency of the PSMA enhancer/promoter by use of adenoviral technology is still unknown. In contrast to nonviral vectors, adenovirus possesses a higher transduction efficiency, a larger capacity for exogenous genes, and a more stable exogenous gene expression (Russell, 2000).
We previously have constructed and analyzed the transcriptional regulatory elements of PSMA and have chosen the best functional element combination of PSMA promoter and enhancer successfully (Zeng et al, 2005). In the present study, we just planned to construct a CD gene that expressed recombinant adenovirus regulated by the PSMA promoter/enhancer and expected to observe the specific cytotoxicity of the Ad-PSMAE-P–CD/5-FC system in prostate adenocarcinomas.
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Materials and Methods |
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Reagents and Plasmids
Plasmid p enhanced green fluorescent protein (EGFP)-PSMAE-P was
constructed previously (Zeng et al,
2005). The recombinant adenovirus vector system was purchased from
Qbiogene. The LA-Taq DNA polymerase kit and reverse
transcriptase–polymerase chain reaction (RT-PCR) reagents were obtained
from TakaRa Biotech (Tokyo, Japan). Restricted endonucleases and other
modified nucleases were from TOYOBO (Osaka, Japan). Transfection reagent was
Lipofectamine2000 (Invitrogen, Carlsbad, Calif). PCR purification and gel
extraction kits were from Omega Bio-tek (Doraville, Ga). The 5-FC and 5-FU
were from Sigma Chemical Co (St Louis, Mo).
Recombinant Adenovirus Vectors
Recombinant replication-defective Ad vectors were prepared, purified, and
titrated according to the manual of the AdEasy vector system (Qbiogene)
(Figure 1). Briefly, the
recombinant adenoviruses were propagated in HEK293 cells and purified by the
continuous CsCl centrifugation method. The purified virus stock was then
dialyzed against 10 mM Tris buffer, pH 7.5, containing 1 mM MgCl2
and 10% glycerol, and the concentrated virus was titrated, aliquoted, and
stored at –80°C. Four recombinant replication-defective Ad vectors
in the experiment were Ad-PSMAE-P–CD, Ad-CMV-CD,
Ad-PSMAE-P-EGFP, and Ad-CMV-EGFP, with the therapeutic toxic gene
E coli CD (GenBank accession number S56903) or a reporter EGFP driven
by a CMV promoter or PSMA promoter (Zeng
et al, 2005). Virus titers were determined by the tissue culture
infectious dose 50 (TCID50) method and OD260 method
according to the manual of the AdEasy vector system (Qbiogene).
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Ad-PSMAE-P–CD/5-FC and Ad-CMV-CD/5-FC Toxicity
Four types of cells, including LNCap, CL-1, A549, and MCF-7, were harvested
from confluent monolayers and plated at 1.5 x 106 to 3
x 106 cells/well in T25 flasks overnight at 37°C. Cells
were infected with various amounts of Ad-CMV-CD or
Ad-PSMAE-P–CD (0–200 PFU/cell). Twenty-four hours after
infection, cells were harvested with 0.25% trypsin/EDTA, plated into 96-well
tissue culture plates at 5000 cells/well in 100 µl of complete media, and
allowed to adhere overnight at 37°C. One hundred microliters of media
supplemented with serial dilutions of 5-FC (6 replicates/dilution) was added,
and cells were further incubated for 2–8 days. Cell survival was
determined by an MTT assay. Fractional cell survival data were plotted against
the logarithm of drug concentration, and 50% infective concentration
(IC50) values were extrapolated by piecewise linear regression;
cell survival status following different end points was also determined.
Bystander Effect of Viral System
CL-1 cells were exposed to Ad-CMV-CD or Ad-PSMAE-P–CD
under conditions that could lead to 100% infection of cells. The infected
cells were then mixed with noninfected cells at a different ratio and
subjected to 5-FC at a concentration of 15 µmol/L. The percentages of
infected cells in the mixtures were 0%,10%,20%,30%,40%, and 100%. Mixed cells
were plated in 6-well plates, and media were changed every 3 days. After 10
days' culture, cells were counted by staining with typan blue.
In Vivo Toxicity of Ad-CD/5-FC System
CL-1 and A549 tumors were established by injecting 1 x 107
cells into 5-week-old athymic BALB/c male mice (Animal Center, Sichuan
University, People's Republic of China), and all cells were inoculated
subcutaneously in the right flank of the mice. Two weeks later, the size of
the tumors reached 8–12 mm in diameter. Xenograft mice were randomly
divided into 6 groups for CL-1 injected mice and 2 groups for A549 injected
mice (5 mice/group) and then treated with recombinant adenoviruses (Ad-CMV-CD
or Ad-PSMAE-P–CD) every 6 days (intratumoral injection, 2
x 109 PFU/0.2 ml). From the second day after adenovirus
injection, mice were further treated with 5-FC (500 mg/kg) or saline every
other day (intraperitoneal injection, every 2 days). Sizes of tumors in each
mouse were measured every 4 days, and volumes were calculated with the
following formula: volume = length x width2 x 0.52
(mm3). Four weeks later, mice were sacrificed. Tissues, including
xenograft tumor, liver, and kidney, were fixed and then stained with
hematoxylin and eosin.
Statistical Analysis
Statistical analysis was performed by SPSS 13.0 software (SPSS Inc,
Chicago, Ill). P < .05 was considered statistically
significant.
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Results |
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Specific Toxicity of Ad-PSMAE-P–CD/5-FC System to Prostate Cancer Cell Line
The 5-FC sensitivity (IC50 in millimoles per liter) varied
according to tumor cell types: 0.238, 0.236, 1.75, and 2.73 mmol/L in LNCap,
CL-1, MCF-7, and A549, respectively. Ad-PSMAE-P–CD/5-FC and
Ad-CMV-CD/5-FC toxicity (5-FC IC50 in millimoles per liter) were
assessed with 4 cell lines after infection with Ad-PSMAE-P–CD
or Ad-CMV-CD at 0, 10, 50, 100, and 200 MOI (PFU/cell). Dose-response curves
from LNCap, CL-1, MCF-7, and A549 were shown in
Figure 4, and a significant
linear relation (P < .01) between Ad-CMV-CD MOI and 5-FC
sensitivity (log-log scale) for each of the 4 cell lines was observed. Similar
results were obtained between Ad-PSMAE-P–CD MOI and 5-FC
sensitivity but only for the LNCap and CL-1 cell lines. The 5-FC sensitivity
of the LNCap and CL-1 cell lines (infected with Ad-CMV-CD at 100 MOI)
increased 314- to 518-fold higher than that of uninfected cells. Although
Ad-PSMAE-P–CD/5-FC could only increase the 5-FC sensitivity
of LNCap and CL-1 by 70- to 134-fold; cytotoxicity induced by
Ad-PSMAE-P–CD was exclusively detected in PSMA-positive
prostate cancer cell lines, which indicated the high specificity of the
suicide gene system.
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In Vitro Effect of Ad-PSMAE-P–CD/5-FC System
Cytotoxicity was examined in vitro by infecting 4 types of cells with
either Ad-PSMAE-P–CD or Ad-CMV-CD at a MOI of 100 added in 15
µmol/L 5-FC (the concentration was selected because higher multiplicities
of infection resulted in the nonspecific killing of cells due to viral
infection [data not shown]). Figure
5 shows that the sensitivity of 4 cells to the pro-drug 5-FC can
obviously be enhanced by infecting them with Ad-CMV-CD in vitro. However,
Ad-PSMAE-P–CD/5-FC could only enhance sensitivity to 5-FC in
PSMA-positive prostate cancer cell lines (LNCap and CL-1). We assumed that
this CD suicide gene therapeutic system was uniquely effective in prostate
cancer cells.
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In Vivo Cytotoxicity
Ad-CMV–CD/5-FC and Ad-PSMAE-P–CD/5-FC injected mice,
which were implanted with CL-1, showed different extents of growth inhibition
ratio (61.5% vs 49.4%), while in A549 implantation groups, the xenograft was
sensitive only to the Ad-CMV–CD/5-FC system (shown in
Figure 8). On day 21 after
treatment, compared to the control groups, the sizes of the tumor in the
Ad-CMV–CD/5-FC and Ad-PSMAE-P–CD/5-FC treated groups
were only 1952 ± 427 mm3 and 2568 ± 487
mm3, respectively. At the same time, in A549 implanted mice, the
volume of tumor could be suppressed only in the Ad-CMV–CD/5-FC treated
group (Table). In
paraffin-embedded tissue sections from sacrificed mice with hematoxylin and
eosin staining indicated, the Ad-CMV–CD/5-FC system showed systemic
toxicity. Necrosis was predominantly observed in the liver tissue, while
necrosis in the kidney was relatively mild. On the other hand, the toxicity of
the Ad-PSMAE-P–CD/5-FC system was confined to the
PSMA-positive tumor cells (Figure
9).
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Discussion |
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As one of the ideal models for gene therapeutic research, several prostate-specific gene promoter elements have been studied, such as PSA, hk2, and probasin. However, it deserves attention that PSA, hk2, or probasin is down-regulated by androgen deprivation (Israeli et al, 1994). Hormone-refractory prostate adenocarcinomas might become insensitized to gene therapeutic systems based on PSA, hk2, and probasin. Prostate-specific membrane antigen is a novel prostate-specific marker in prostate adenocarcinoma, which was first identified in the frozen tissue of prostate cancer early in 1987. The PSMA expression level in the prostate is 1000-fold higher than that in nonprostate tissues. Further studies have demonstrated that PSMA expression in advanced or metastatic prostate adenocarcinomas is several times higher than in nonmalignant prostate tissues (Israeli et al, 1994; Silver et al, 1997). It is exciting that PSMA expression is up-regulated by androgen deprivation (Wright et al, 1996), which indicates that PSMA is an excellent target gene in the therapy of hormone-refractory prostate adenocarcinomas. Early in 2000, O'Keefe et al utilized PSMA promoter and enhancer to regulate CD gene expression mediated by plasmids, and their result showed the specific expression and conversion potential of CD controlled by PSMA regulatory elements (O'Keefe et al, 2000; Uchida et al, 2001). Lee et al (2002) constructed a novel prostate-specific promoter PSES derived from the PSA and PSMA promoter/enhancer, which produced high selective activity in prostate cancer cells. The infectivity of tumor cells transfected with nonviral vectors is lower than that of viral vectors; lower transduction efficacy leads to a weaker therapeutic effect. The effect of CD-produced adenovirus mediated by PSMA regulatory elements is still unclear and deserves examination.
In the past 2 years, our group has screened and chosen the best regulatory element combination of PSMA enhancer and promoter (Zeng et al, 2005). We first constructed CD-produced and reporter gene (EGFP)-produced recombinant adenoviruses controlled by the prostate-specific regulatory element chosen previously. EGFP expression in vitro appeared exclusively in PSMA-positive prostate cancer cell lines when cells were infected with Ad-PSMAE-P–EGFP, demonstrating the specific regulatory function of the PSMA enhancer/promoter repeatedly. Only LNCap and CL-1 cells were sensitized to the Ad-PSMAE-P–CD/5-FC system in the 4 examined cells, while the Ad-CMV–CD/5-FC system showed its versatility to all cell lines. The sensitivity of 5-FC to LNCap and CL-1 cells infected with Ad-PSMAE-P–CD predominantly increased 70- to 134-fold higher than that of uninfected cells. The CL-1 cell line, derived from LNCap, was one of the androgen-independent prostate cancer cell lines, which also expressed PSMA. In the present study, the results demonstrated that the Ad-PSMAE-P–CD/5-FC system could lead to cytotoxicity in PSMA-positive cells (LNCap and CL-1), whether it was androgen-dependent or not. Although we did not compare the effect of Ad-PSMAE-P–CD with the PSMAE-P-based CD gene-expressed plasmid parallel in our experiment, compared with data from O'Keefe et al (2000), the transduction efficiency of adenovirus was definitively better than that of plasmids. According to statistic analysis, we found that the 4 tested cells maintained different sensitivities to the Ad-CMV–CD/5-FC system, which may be because of different densities of coxsackievirus and adenovirus receptor upon the extracellular surface (Kraaij et al, 2005). Compared with the CMV promoter, the regulatory activity of the PSMA enhancer/promoter was indeed lower, whereas Ad-PSMAE-P–CD/5-FC could actually achieve targeting cytotoxicity to PSMA-positive cancer cells.
In addition, the flow cytometry assay confirmed that cell arrest before the S phase was involved in the growth inhibition of LNCap or CL-1 cells treated with Ad-PSMAE-P–CD or Ad-CMV–CD plus 5-FC. The ratio of cell arrest before S phase in LNCap cells infected with Ad-PSMAE-P–CD was lower than that of LNCap cells infected with Ad-CMV–CD (69.9% vs 87%); however, it did not appear to affect the cytotoxicity of the Ad-PSMAE-P–CD/5-FC system in PSMA-positive cells. Although the adenoviral vector has a higher infectivity than the plasmid vector, it was impossible to infect 100% of the tumor cells. In the present study, the bystander effect of adenovirus was also observed; the results verified the significant effect of this kind of phenomenon beyond doubt. The present study confirmed the specific cytotoxic effects of the Ad-PSMAE-P–CD/5-FC system in vivo, and the tumor growth of mice implanted with CL-1 cells decreased almost 50% during the observation time. Compared with the systemic toxicity of the Ad-CMV–CD/5-FC system, the Ad-PSMAE-P–CD/5-FC system exhibited its toxicity only in PSMA-positive tumor cells without adverse results to other organs, such as the liver and kidney. In brief, the Ad-PSMAE-P–CD/5-FC system could effectively delay tumor growth in vivo without systemic toxicity.
Our experimental research confirmed the specific transcriptional activity of the PSMA enhancer/promoter in vivo and in vitro, and we first constructed Ad-PSMAE-P–CD/5-FC system. The transduction efficiency of the recombinant adenovirus might be better than the general plasmid vector in targeting gene therapy. PSMA is worth being investigated in depth in gene therapy for prostate adenocarcinoma.
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Footnotes |
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Present address: Department of Urology, West China Hospital, Sichuan
University, 37 Guoxue Xiang St, Chendu, Sichuan, 610041, People's Republic of
China. 
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represents CL-1 treated with Ad-PSMAE-P-CD or Ad-CMV-CD plus 5-FC;
represents A549 treated with Ad-PSMAE-P-CD or Ad-CMV-CD
plus 5-FC; 
represents MCF-7 treated with Ad-PSMAE-P-CD or
Ad-CMV-CD plus 5-FC; 
represents CL-1 treated with
Ad-PSMAE-P-CD or Ad-CMV-CD alone; 
represents CL-1 treated
with 5-FC alone.
